This is a proposal to investigate the role of tissue boundaries in cranial suture development and the pathophysiology of craniosynostosis. More broadly, this proposal focuses on how boundaries control pattern in a complex, multicomponent structure. Our recent results on the mechanism of Saethre-Chotzen syndrome, caused by heterozygous loss of function of Twist1, demonstrated that Twist1 mutant mice have a deficiency in the neural crest-mesoderm boundary at the coronal suture. The boundary normally lies between the mesoderm-derived cells of the prospective suture and the neural crest derived osteogenic cells of the prospective frontal bone. We showed that ephrin-Eph signaling, controlled by Twist1, has a role in the maintenance of this boundary: EphA4 is expressed in a layer of cells ectocranial to the prospective bone, through which osteogenic precursor cells migrate. Reduced dosage of Twist1 and EphA4 results in inappropriate targeting of migratory osteogenic precursor (MOP) cells to the coronal suture. This pathfinding defect, we proposed, is a key cause of craniosynostosis in Twist1 and EphA4 mutants. In work now under submission, we found that the Notch ligand, Jagged1, is expressed in a layer of cells in the coronal suture that demarcate the osteogenic-non-osteogenic boundary. Expression of Jagged1 is markedly reduced in such cells in Twist1 mutants. Moreover, conditional inactivation of Jagged1 in these cells results in synostosis, and to an upregulation of Notch2 and Hes1 in the suture. These results are the basis of our three-part overall hypothesis that Twist1 is at a node a regulatory hierarchy, controlling ephrin-Eph and Jagged1/Notch signaling, that Ephrin-Eph signaling functions primarily in the ectocranial mesenchyme to control MOP cell migration, and that Jagged1 functions in sutural mesoderm in the specification of border cells within the suture. To test this hypothesis, we propose first to determine whether the MOP cell targeting defect is inherent in MOP cells or is a result of a change in the ectocranial layer through which MOP cells migrate or of the sutural mesenchyme that they invade. We will approach this using conditional targeting and an array of Cre mice. Second, we will ask whether preventing the expansion of Notch2 and beta catenin expression in sutural cells mitigates the craniosynostosis phenotype, and whether forcing expression of Notch2 in sutural cells causes synostosis. Finally, we will use gene profiling to test the hypothesis that a change in the identity of cells of the coronal suture is the first event in synostosis, and that it is followed by-and exacerbated by-a defect in the targeting of osteogenic precursor cells. PUBLIC HEALTH RELEVANCE: This is a proposal to study how cranial sutures form and how three genes-Twist1, EphA4 and Jagged1- control the development of specific cells within the sutures. These genes are of special interest because humans with mutations in them have birth defects that affect their skulls. By studying how these genes work, we will learn more about basic processes of skull development as well as how mutations in these genes lead to birth defects. In the long term, this work may help to devise new treatments of such birth defects.